In This Issue

Francisella Foils Apoptosis

Francisella tularensis is a highly virulent human pathogen that is the causative agent of tularemia. Interactions between neutrophils and F. tularensis are known to be important in disease pathogenesis, but are not well understood. Schwartz et al. (p. 3351) investigated the effects of F. tularensis on neutrophil lifespan, extending previous data demonstrating its ability to disrupt neutrophil oxidant production. The authors hypothesized that F. tularensis interferes with the constitutive spontaneous apoptosis of neutrophils that is necessary for the proper resolution of inflammation. Indeed, infection of human neutrophils with F. tularensis live vaccine strain or virulent strain Schu S4 significantly delayed apoptosis compared with controls. The bacteria impaired apoptosis by both the extrinsic and intrinsic pathways, as demonstrated by significantly reduced activation of caspases-3, -8, and -9, as well as strongly reduced Fas-triggered apoptosis. This interference with apoptosis required live bacteria, but did not strictly require cell contact between pathogen and neutrophils, nor did it require bacterial LPS or capsular polysaccharides. By demonstrating inhibition of neutrophil apoptosis, these data provide insight into tularemia pathogenesis that may support future efforts to combat this disease.

NK Cell Calculations

IL-15 drives NK cell proliferation during viral infections and is also important in NK cell development, survival, and homeostasis. To more thoroughly understand the control of NK cell expansion, Zhao and French (p. 2981) developed two-compartment mathematical models addressing the kinetic parameters involved in NK cell proliferation and the influence of IL-15 on this process. The two compartments in these models represented dividing and quiescent NK cell subpopulations. The models were developed based on in vitro proliferation of NK cells in response to different concentrations of IL-15, and their validity was then assessed in predicting facets of IL-15–stimulated NK cell proliferation. One of the models was found to accurately predict NK cell numbers at different time points, and this model indicated that the quiescent and dividing NK cells had distinct proliferation and cell death kinetics. Differing concentrations of IL-15 had a greater impact on the NK cell division rate and the recruitment of quiescent cells into the dividing compartment than on the NK cell death rate. The validity of the model was tested, and upheld, under both stable and changing IL-15 concentrations, suggesting its utility in future studies of NK cell responses in vivo. This model provides insight into the dynamics of NK cell expansion, and the ability to predict NK cell activity mathematically has the potential for translational application.

GM-CSF Touches a Nerve

Although Th1 and Th17 cells mediate neuroinflammation in experimental autoimmune encephalomyelitis (EAE), their signature cytokines, IFN-γ and IL-17, have been found to be dispensable for disease development. Instead, GM-CSF appears to be the key cytokine responsible for demyelinating disease. In this issue, Lukens et al. (p. 3107) sought to identify the cells and mechanisms responsible for GM-CSF production during EAE. In vitro, IL-1R was required for GM-CSF production by both αβ and γδ T cells in response to stimulation with anti-CD3 or cytokines. During EAE induction, GM-CSF–producing T cells were heterogeneous and included cells that also produced IFN-γ, IL-17, or both, as well as cells that produced GM-CSF but did not secrete additional Th cell cytokines. γδ T cells were found to be another major source of GM-CSF, supporting the involvement of these cells in EAE pathogenesis. During the effector phase of EAE, IL-1R deficiency prevented GM-CSF production in the CNS and greatly ameliorated disease. Inflammasome-mediated generation of IL-1β upstream of IL-1R and MyD88 activity downstream of IL-1R were important for GM-CSF production by T cells. By clarifying how the critical cytokine GM-CSF is generated during neuroinflammation, these results promote the understanding of autoimmune disease progression.

Exhausting Immunotherapy

The anti-CD20 mAbs ofatumumab (OFA) and rituximab (RTX) enlist host mechanisms, such as complement-dependent cytotoxicity, to target B cells in cancer treatment. In a phase II trial of OFA infusion combined with chemotherapy for the treatment of chronic lymphocytic leukemia (CLL), Beurskens et al. (p. 3532) investigated the effects of OFA on complement-mediated killing of circulating B cells. Treatment significantly reduced B cell numbers and complement levels, but spared a number of CLL B cells that continued to circulate with complement component C3d covalently bound to their surfaces. In vitro experiments demonstrated that a low dose of OFA could induce maximal cytotoxicity and that higher doses of either OFA or RTX resulted in complement depletion. These data suggest that the potential for complement-dependent cytotoxicity of tumor cells is saturable and is strongly influenced by the dose of opsonizing mAb, such that a high dose of mAb may lead to complement exhaustion and ineffective killing in a secondary tumor challenge. Whereas drugs are routinely used in tumor therapies at the maximal tolerated dose, this study suggests that lower, more frequent doses of mAbs that employ complement-mediated effector functions may result in more effective tumor immunotherapy.

Giving Flu Vaccines a Boost

Subunit influenza virus vaccines are safer but less immunogenic than whole-virus vaccines. Caproni et al. (p. 3088) analyzed the innate immune signatures generated by several adjuvants and their ability to boost the immunogenicity of a trivalent influenza subunit vaccine in mice. Of TLR-independent (MF59 and alum) and TLR-dependent (CpG, resiquimod, and Pam3CSF4) adjuvants, only MF59 and Pam3CSF4 boosted influenza-specific IgG responses, although all adjuvants boosted tetanus toxoid-specific IgG. MF59, an oil-in-water emulsion, could also enhance influenza-specific splenic CD4+ T cell responses. Adjuvant-induced gene expression profiles were analyzed in vitro in murine splenocytes and in vivo both in the muscle (the vaccine injection site) and the draining lymph nodes (LNs). Although MF59 did not strongly modulate gene expression in vitro or in the LNs, it activated transcription of a large number of genes involved in inflammation and cellular migration at the injection site, which were associated with the recruitment of CD11b+ cells to this site. Despite their adjuvanticity, MF59 and Pam3CSF4 did not modulate IFN-related genes. The TLR7 agonist resiquimod was the strongest modulator of gene expression in the LNs and was the only adjuvant to induce IFN responses; however, it was unable to boost flu vaccine adjuvanticity. These data regarding the mechanistic basis of adjuvant activity may contribute to the development of better flu vaccines.

Marked as Myeloid

Controversy has surrounded the lineage potential of the earliest thymic progenitors (ETPs), as studies have suggested that ETPs are either able or unable to differentiate into myeloid lineage cells. In hopes of resolving this debate, Haymaker et al. (p. 3208) generated tools to trace myeloid cell progenitors by the expression of IL-13Rα1. Within the thymus, IL-13Rα1 expression was observed only on a subpopulation of DN1 double-negative thymocytes. These IL-13Rα1+ ETPs were found to be committed to the myeloid lineage with no ability to mature into lymphoid cells in vitro or in vivo. ETPs from IL-13Rα1–deficient mice were unable to differentiate into myeloid cells but showed no defects in lymphoid differentiation, indicating that IL-13Rα1 was necessary for myeloid cell maturation. Adoptive transfer experiments suggested that IL-13Rα1+ ETPs derived from lineage-negative progenitors that migrated from the bone marrow to the thymus rather than from multipotent IL-13Rα1− thymic progenitors. Upon maturation, these thymic myeloid cells remained in the thymus and were found to have Ag presentation capabilities. Taken together, these data suggest that ETPs consist of distinct populations that are able to give rise to either lymphoid or myeloid cells and are separable on the basis of IL-13Rα1 expression.

Supporting Survival

The widely expressed intracellular protein calcium-modulating cyclophilin ligand (CAML) is necessary for proper T cell development. Although CAML is known to interact with the B cell-specific TNFR family member TACI, its function in mature B cells remains unknown. To address this issue, Zane et al. (p. 3009) generated B cell-specific CAML-deficient mice (bCAML−/−) and inducible CAML knockout mice (eCAML−/−). Compared with wild-type mice, bCAML−/− mice had reduced numbers of mature B cells, both in the spleen and peritoneum, with a strikingly large decrease in follicular B cells. CAML-deficient B cells displayed an activated phenotype and defective survival that was associated with an accelerated turnover rate in the spleen. BCR signaling was not impaired in bCAML−/− mice, and the survival defect did not appear to involve the B cell survival factor BAFF. Analysis of eCAML−/− mice demonstrated that CAML was not necessary during B cell development but was required instead for the long-term survival of mature B cells. Adoptive transfer experiments confirmed the survival defect of both bCAML−/− and eCAML−/− cells. Thus, there is an ongoing requirement for CAML to support the survival of mature B cells, especially follicular B cells, under steady-state conditions.

A Healthy Dose of Symbiosis

Insects, like humans, harbor symbiotic bacteria that may influence immunity. In this issue, Weiss et al. (p. 3395) analyzed the development of the immune system in tsetse flies (Glossina species) in the absence of symbiotic bacteria. These aposymbiotic tsetse flies (Glossina morsitans morsitans [GmmApo]) underwent intrauterine development in mothers lacking all three of their normal symbionts. Compared with wild-type flies, GmmApo flies had very few circulating phagocytic hemocytes and were dramatically more susceptible to E. coli infection. GmmApo flies also showed downregulation of genes associated with cellular immunity and upregulation of genes associated with humoral immunity. Transfer of hemocytes from mature wild-type, but not GmmApo, flies could restore the immune response to E. coli infection in aposymbiotic flies. In addition, symbiont-depleted mother flies that were fed cellular extracts of the symbiont Wigglesworthia produced offspring (GmmApo/Wgm) that had increased numbers of hemocytes and upregulated transcription factors necessary for hematopoiesis, compared with the offspring of unsupplemented mothers. These GmmApo/Wgm flies also regained resistance to E. coli challenge. By demonstrating that the obligate symbiont Wigglesworthia is necessary for proper immune system development in tsetse flies, these data provide an interesting example of the importance of the host microbiota in immunity.